Exposures to environmental agents can have an effect on mammary gland development and may affect breast cancer risk. This project explores the mechanisms active during the window of susceptibility during adolescence. By defining the molecular architecture of the developing and changing mammary gland over the lifespan we will be able to develop new and improved animal models and biomarkers to study the impact of environmental stressors on breast cancer and elucidate the effects of timing of these exposures during critical windows of vulnerability. This project will use state-of-the-art imaging, proteomic and genetic tools to determine when, which and how environmental insults regulate cell behavior in the mouse mammary gland during development in vivo, and in conversion of mammary epithelial cells to premalignant phenotypes. Experiments will be conducted cooperatively, using both normal and tumor prone mouse models to characterize pathways related to breast development during early life, puberty, pregnancy and ageing, and to determine how they are affected by exposures to environmental stressors occurring at different windows of vulnerability to environmental stressors. These studies in mice will be complemented using human mammary epithelial cells in culture to develop new assays that can assess how exogenous agents alter their ability to overcome finite lifespan and constraints imposed by intercellular interactions. By defining critical windows of vulnerability, and creating improved animal and human cell culture models that will lead to identification of biomarkers, these studies have the potential for translation to women to determine the impact of environmental stressors on breast cancer. We will determine the alterations in the mammary microenvironment and cells in vivo. We hypothesize that the characteristics and regulation of putative mammary stem/progenitor ceils are critical determinants of cancer susceptibility and will study this population using fluorescent marked reporter populations. We will then determine the effects of exposure to prototypical environmental stressors during prenatal development, pubertal branching morphogenesis, pregnancy and aging on the mammary gland in normal and cancer prone mice in vivo. We will use in vitro mechanism-based screens to detect agents that possess signatures indicative of possible mammary gland carcinogenicity. An essential feature of Collaborative Project 1 is regular bi-directional communication with the epidemiological studies in Collaborative Project 2 and the Community Outreach and Translational Core to participate in the selection of appropriate environmental stressors and to develop biomarkers.